Labeled oligonucleotides are widely used probes for detecting specific macromolecule targets such as nucleic acids and proteins. Their ability to bind targets with high specificity has rendered them useful in in vitro assays such as polymerase chain reaction (PCR) protocols. However, delivering these types of target specific probes into living cells remains a major challenge as cells are both naturally resistant to nucleic acid uptake and contain a variety of pathways to remove these foreign genetic materials. Thus, methods that deliver these materials into cells, in a manner in which they retain both their specific binding properties and fluorescent signaling ability are of great interest.
The discovery and subsequent development of the oligonucleotide-nanoparticle conjugate have lead to a variety of new opportunities in molecular diagnostics (Elghanian et al., 1997, Science 277: 1078-1081; Nam et al., 2003, Science 308: 1884-1886) and materials design (Mirkin et al., 1996, Nature 382: 607-609; Alivisatos et al., 1996, Nature 382: 609-611; Demers et al., 2003, Angew. Chem. Int. Ed. 40: 3071-3073). Recently, it has been demonstrated that oligonucleotide-functionalized nanoparticles enter cells and act as antisense agents to control gene expression (Rosi et al., 2006, Science 312: 1027-1030). These “antisense particles” are not simply delivery vehicles (Sandhu et al., 2002, Bioconjugate Chem. 13: 3-6; Tkachenko et al., 2003, J. Am. Chem. Soc. 125: 4700-4701), but rather single entity regulation and transfection agents that undergo cellular internalization, resist enzymatic degradation, and bind intracellular targets with affinity constants that are as much as two orders of magnitude greater than free oligonucleotides (Lytton-Jean and Mirkin, 2005, J. Am. Chem. Soc. 127: 12754-12755). Moreover, they can be easily modified with potent, i.e., highly stable, designer materials such as locked nucleic acids (Seferos et al., 2007, Chem Bio Chem 8: 1230-1232) and are nontoxic under conditions required for gene regulation. Indeed, it has been shown that, unlike oligonucleotides free in solution, oligonucleotide-modified gold nanoparticles are readily taken up by cells in high numbers. This property has lead to the discovery that oligonucleotide-modified gold nanoparticles can be used as agents for intracellular gene control, where they provide rapid intracellular delivery of DNA, and further increase the efficacy of the oligonucleotides in the cells based on cooperative properties. These oligonucleotide functionalized nanoparticles have been shown to enter a variety of cell types, and can be used to introduce high local concentrations of oligonucleotides.
It has also previously been shown that gold nanoparticles that are densely functionalized with DNA bind complementary DNA in a highly cooperative manner, resulting in a binding strength that is two orders-of-magnitude greater than that determined for analogous DNA strands that are not attached to a gold nanoparticle. This property has rendered nanoparticles particularly useful for DNA and protein diagnostic assays in addition to those uses described above.
One class of oligonucleotides of interest are those that can detect a specific target with a recognition sequence. These types of structures, if introduced into living cells, are especially of interest for medical diagnosis, drug discovery, and developmental and molecular biology application. However, current delivery/transfection strategies lack the attributes required for their use such as 1) low toxicity, 2) high cellular uptake, and 3) provide resistance to enzymes that lead to false positive signals.
Probes to visualize and detect intracellular RNA including those used for in situ staining (Femino et al., Science 280: 585-590, 1998; Kloosterman et al., Nat. Methods 3: 27-29, 2006), molecular beacons (Tyagi et al., 1996, Nat. Biotechnol. 14: 303-308; Sokol et al., 1998, Proc. Natl. Acad. Sci. USA 95: 11538-11543; Peng et al., 2005, Cancer Res. 65: 1909-1917; Perlette et al., 2001, Anal. Chem. 73: 5544-5550; Nitin et al., 2004, Nucleic Acids Res. 32: e58), and FRET-pairs (Santangelo et al., 2004, Nucleic Acids Res. 32: e57; Bratu et al., 2003, Proc. Natl. Acad. Sci. USA 100: 3308-13313) each of which are important biological tools to measure and quantify activity in living systems in response to external stimuli (Santangelo et al., 2006, Annals of Biomedical Engineering 34: 39-50). However, the delivery of oligonucleotide-based reporters into cellular media and cells has proven to be a major challenge for intracellular detection. The cellular internalization of oligonucleotide-based probes typically requires transfection agents such as lipids (Zabner et al., 1995, J. Bio. Chem. 270: 18997-19007) or dendrimers (Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci. USA 93: 4897-4902) which can be toxic or alter cellular processes. Furthermore, oligonucleotides are prone to degradation within cells (Opalinska and Gewirtz, 2002, Nat. Rev. Drug Disc. 1: 503-514), and in the case of fluorophore-labeled probes, this can lead to a high background signal that is indistinguishable from a true recognition event (Li et al., 2004, Nucleic Acids Res. 28: e52; Rizzo et al., 2002, Molecular and Cellular Probes 16: 277-283).
Accordingly, while nanoparticle have been designed that can recognize targets with a high degree of specificity, it is difficult to detect a positive effect arising from the specific interaction, particularly with the sensitivity to detect such an interaction at the single cell level.
Thus there exists a need in the art to develop materials which are capable of entering a cell to associate with a specific target and methods to detect and quantitate the resulting intracellular interaction.